Inhibition of tumor angiogenesis has emerged as a promising strategy to treat both primary and metastatic tumors. Although effective at shrinking tumors, antiangiogenic agents are not tumoricidal, and tumor regrowth frequently occurs after termination of treatment with the angiogenesis inhibitor. One strategy to overcome this limitation is to combine antiangiogenic agents with conventional therapies such as radiotherapy, although the underlying physiological rationale for such combinations remains unexplored. The primary focus of this proposal is to explore both physiological and molecular changes following both antiangiogenic and angiogenic stimuli. Although established, transplantable murine tumor models have long been the standard for radiobiological investigations, the dependence of physiological function on tumor derivation remains uncertain. Differences in vascular development, specifically in regards to relative proportions of host versus tumor vessels, could be vital in the evaluation of antiangiogenic and combined modality, preclinical strategies. The current application proposes to examine tumor microregional changes in vascular structure and hypoxia, in conjunction with alterations in angiogenic and antiangiogenic growth factor expression, among a panel of carefully chosen murine tumor models. First, spontaneous mammary carcinomas will be contrasted with slow and fast growing 1st generation transplants of these tumors. Second, nonmetastatic MCa-IV versus aggressive, metastatic MCa-35 established mammary carcinoma models will be studied. Our methods include: a) cryospectrophotometry to define intravascular oxygen levels, b) immunohistochemical staining to quantify total and perfused vessels, tumor cell proliferation, apoptosis, and percent necrosis, c) immunohistochemistry, in situ hybridization, and RNA protection assays to quantify growth factor and receptor levels, d) hypoxic marker uptake (EF5) to delineate regional changes in oxygenation, and e) growth delay assays to evaluate response to single and multi-fraction radiotherapy, as well as antiangiogenic agents. In view of an increasing interest in the use of multi- modality therapies that incorporate potentially angiogenic and antiangiogenic agents, this proposal will quantitate the effects of both angiogenic growth factors (VEGF and FGF2) and antiangiogenic agents (endostatin). Endostatin will also be combined with radiotherapy to determine whether the radiation is potentiated by the antiangiogenic agent. In summary, our primary goals are to determine the fundamental relationships between molecular and pathophysiological changes in disparate tumor models and whether observed differences can be therapeutically exploited.